GATA3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in breast cancer

Background Inadequate DNA damage repair promotes aberrant differentiation of mammary epithelial cells. Mammary luminal cell fate is mainly determined by a few transcription factors including GATA3. We previously reported that GATA3 functions downstream of BRCA1 to suppress aberrant differentiation in breast cancer. How GATA3 impacts DNA damage repair preventing aberrant cell differentiation in breast cancer remains elusive. We previously demonstrated that loss of p18, a cell cycle inhibitor, in mice induces luminal-type mammary tumors, whereas depletion of either Brca1 or Gata3 in p18 null mice leads to basal-like breast cancers (BLBCs) with activation of epithelial-mesenchymal transition (EMT). We took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis. Results Depletion of Gata3, like that of Brca1, promoted DNA damage accumulation in breast cancer cells in vitro and in basal-like breast cancers in vivo. Reconstitution of Gata3 improved DNA damage repair in Brca1-deficient mammary tumorigenesis. Overexpression of GATA3 promoted homologous recombination (HR)-mediated DNA damage repair and restored HR efficiency of BRCA1-deficient cells. Depletion of Gata3 sensitized tumor cells to PARP inhibitor (PARPi), and reconstitution of Gata3 enhanced resistance of Brca1-deficient tumor cells to PARP inhibitor. Conclusions These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression. Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-024-01881-6.


Background
The integrity of the mammalian genome is under constant assault from external factors such as UV light and internal causes such as errors in DNA replication.DNA damage that is unrepaired or improperly repaired results in chromosomal rearrangement and, ultimately, cancer.DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage in promoting genome instability and tumorigenesis.DNA DSBs are mainly repaired through homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways.Defects in these pathways are closely associated with the development and progression of cancers, particularly hormone-related cancers including breast cancer [1,2].Recently, it has been reported that inadequate DNA damage repair promotes aberrant mammary epithelial cell differentiation, leading to breast cancer [3,4].How deficiency in DNA damage repair promotes breast cancer development and progression remains elusive.
Mammary epithelia are mainly composed of luminal and basal cells.The determination and maintenance of luminal cell fate is orchestrated by networks of transcription factors, including BRCA1 and GATA3 [5,6].Breast cancer comprises of, among others, two main subtypes: estrogen receptor (ER)-positive luminal and ER-negative basal-like [7].Luminal-type tumors respond to hormone therapies.Basal-like breast cancers (BLBCs) likely originate from luminal progenitors and are poorly differentiated and aggressive [8,9].In addition to functioning as a transcription factor, BRCA1 is also a tumor suppressor directly involved in the repair of DNA DSBs and the maintenance of genomic stability.Functional loss of BRCA1 by germline or somatic mutation, or promoter methylation is associated with more than half of BLBCs [12][13][14].BRCA1-deficient cancer cells harbor a deficiency of DNA repair by HR, which makes these cells respond well to poly ADP ribose polymerase (PARP) inhibitors.We and others have demonstrated that depletion of Brca1 in mice activates epithelial-to-mesenchymal transition (EMT) program and induces highly heterogeneous BLBCs [4,[10][11][12].Interestingly, depletion of BRCA1 or its interacting proteins including FANCD2, BRG1, NUMB, and HES1 in immortalized mammary epithelial cells impairs DNA damage repair promoting aberrant luminal to basal and mesenchymal differentiation [3,4].However, deficiency of BRCA2, a BRCA1-interacting protein and also functions in DNA damage repair, does not cause aberrant differentiation [4].It is poorly understood whether and how additional BRCA1-interacting proteins control DNA damage repair and differentiation in breast cancer development and progression.
GATA3, a BRCA1-interacting protein, plays a critical role in the development of multiple organs and cell lineages including the mammary gland [13][14][15][16][17][18][19].In human beings, germline mutations of GATA3 are associated with congenital hypoparathyroidism-deafness-renal disease (HDR) syndrome [20,21], and somatic mutations of GATA3 have been detected in ~ 15% of breast cancers [7,22].Expression of GATA3 is a key feature of luminal breast cancers, whereas the GATA3 gene is often silenced by DNA methylation [23,24] and its expression is barely detectable in BLBCs [6,25].In mice, germline or epithelium-specific deletion of Gata3 causes early lethality or severe growth defects [13][14][15]26].Loss of Gata3 in transgenic mice stimulates mammary luminal tumor progression [27,28] and overexpression of GATA3 suppresses epithelial-mesenchymal transition (EMT) in cancer cell lines [29,30].We discovered that GATA3 functions downstream of BRCA1 to promote aberrant luminal to basal and mesenchymal differentiation in the induction of BLBCs [31,32].It has recently been reported that depletion of GATA3 may impair HR-mediated DNA damage repair by downregulating CtIP in breast cancer cells in vitro [33].However, whether GATA3 contributes to DSB repair in mammary tumorigenesis and progression and whether GATA3 functions downstream of BRCA1 to do so remain elusive.
Depletion of either Brca1 or Gata3 in mice impairs the proliferation of mammary epithelial cells (MECs) with induction of cell cycle inhibitors preventing us from direct investigation of their functional loss in DNA damage and dedifferentiation in mammary tumorigenesis [12,25,31,32,34].We previously demonstrated that in mice loss of p18Ink4c (p18), an INK4 family cell cycle inhibitor, promotes MEC proliferation and induces Gata3 and Brca1-proficient luminal type mammary tumors, whereas depletion of either Brca1 or Gata3 on top of p18 null background leads to the development of BLBCs with activation of EMT [12,25,31,32,35].In the present study, we took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis.

Haploid loss of Gata3 or Brca1 in p18 null mice promotes DNA damage accumulation in induction of BLBCs with EMT features
Inspired by our finding that GATA3 functions downstream of BRCA1 to suppress aberrant luminal to basal and mesenchymal differentiation in the induction of BLBCs [31,32], and the discovery that inadequate DNA damage repair promotes aberrant mammary epithelial cell differentiation [3], we hypothesized that GATA3, like BRCA1, also functions in DNA repair during mammary tumorigenesis.To test this hypothesis, we performed IHC analysis with the antibody against γH2AX, a marker for DNA DSBs, in mammary tumors spontaneously developed in p18 −/− and p18 −/− ;Gata3 +/− mice.We found that γH2AX-positive cells were barely detectable in p18 −/− tumors; however, significantly more γH2AXpositive cells were found in p18 −/− ;Gata3 +/− tumors (Fig. 1a, b).In line with our previous finding, we confirmed that p18 −/− mammary tumors were well-differentiated luminal type and that p18 −/− ;Gata3 +/− tumors were poorly differentiated basal-like with EMT features (Fig. 1c, and details in [31,32]).These results indicate that deficiency of Gata3 promotes DNA damage accumulation and induces basal and mesenchymal differentiation in mammary tumorigenesis.As a direct comparison, we also performed a similar analysis for mammary tumors spontaneously developed in p18 −/− ;Brca1 +/− mice.As expected, more γH2AX-positive cells were observed in p18 −/− ;Brca1 +/− tumors than in p18 −/− tumors (Fig. 1a, b).Notably, the number of γH2AX-positive cells in p18 −/− ;Gata3 +/− tumors was less than that in p18 −/− ;Brca1 +/− tumors, suggesting that the efficiency of DNA damage repair by GATA3 is not as strong as that by BRCA1.We determined tumor cell proliferation in vivo and observed that the percentages of Ki-67-positive cells were comparable in p18 −/− and p18 −/− ; Brca1 +/− or p18 −/− ; Gata3 +/− tumors (Fig. 1d, Additional file 1: Fig. S1).These results are consistent with our previous discovery that loss of p18 rescues Brca1-or Gata3-deficient mammary cell proliferation [31,35], and also suggest that GATA3 deficiency promoted γH2AX-focus formation in vivo is less likely due to cell cycle arrest.Together, these results indicate that a deficiency of Gata3, like that of Brca1, impairs DNA damage repair and promotes aberrant basal and mesenchymal differentiation in mammary tumorigenesis and progression.
We then performed IHC for p18 −/− ;Gata3 +/− mammary tumors and found that the expression of GATA3 was highly heterogeneous, some of the cells expressed GATA3, and some did not (Fig. 1e).We analyzed genomic DNA isolated from tumors and observed that the remaining wild-type Gata3 allele was retained in all three p18 −/− ;Gata3 +/− mammary tumors tested (Fig. 1f ).Given that the remaining wild-type Brca1 allele was lost in CK5-positive basal-like cells, not in CK5-negative cells, in Brca1 heterozygous mammary tumors [35], and that GATA3 functions downstream of BRCA1 to suppress breast cancer [32], it will be interesting to determine if LOH of Gata3 occurs in certain groups of Gata3 heterozygous mammary tumor cells.

Depletion of Gata3 in luminal tumor cells promotes DNA damage accumulation and produces BLBCs
Taking advantage of the luminal type mammary tumor model system we established [31,32], we directly test if Gata3 deficiency impairs DNA damage repair in luminal tumor cells in vivo.We knocked down Gata3 in MMTV-PyMT tumor cells that were isolated and screened from MMTV-PyMT mammary tumors and were confirmed as Gata3 and Brca1 proficient (Gata3 +/+ ;Brca1 +/+ ) luminal type before and after transplantation into mammary fat pads (MFPs) of recipient mice (Fig. 2a, and details in [31,32]).We transplanted MMTV-PyMT luminal tumor cells into MFPs of mice and performed IHC analysis for newly generated mammary tumors.We found that the tumors generated by Gata3-depleted cells displayed significantly more γH2AX-positive cells than tumors generated by control cells (Fig. 2b, c, Additional file 1: Fig. S2).Together with our previous finding that depletion of Gata3 in these luminal tumor cells promotes basal-like differentiation in tumors newly generated [31], these data demonstrate that depletion of Gata3 in luminal tumor cells also impairs DNA damage repair and promotes basal-like differentiation.

Depletion of GATA3 in breast cancer cells impairs DNA damage response in vitro
It has been reported that depletion of GATA3 in MCF7 cells reduces the expression of CtIP and impairs etoposide-induced DNA damage repair and that the overexpression of WT GATA3, but not the overexpression of a few mutants including D335E, promotes HR [33].However, since MCF7 cells harbor a heterozygous mutant GATA3 D335E allele and express a barely detectable WT GATA3 protein [22,32,36], whether the loss of function of WT GATA3 impacts DNA damage repair remains to be confirmed.We knocked down GATA3 in T47D cells expressing WT GATA3 and harboring a mutant p18 [36,37] and noticed that depletion of GATA3 downregulated the expression of CtIP and upregulated the expression of γH2AX (Fig. 3a, Additional file 1: Fig. S3).We also checked the expression of RNF-8 and Rad51, both of which play a critical role in DSB repair.We found that the knockdown of GATA3 slightly reduced the expression of RNF-8 and enhanced that of Rad51 Fig. 1 Gata3-and Brca1-deficient mammary tumors display significantly enhanced number of γH2AX-positive tumor cells, and the remaining wild-type Gata3 allele is retained in Gata3 heterozygous tumors.a Representative IHC analysis of mammary tumors spontaneously developed in p18 −/− ;Gata3 +/− , p18 −/− ;Brca1 +/− , and p18 −/− mice.Typical γH2AX-positive tumor cells are indicated.The inset shows the enlarged γH2AX-positive cells.b The H-scores for γH2AX in a were calculated.The results represent the mean ± SD of five individual tumors per group.The asterisk (*) denotes a statistical significance from p18 −/− and p18 −/− ;Gata3 +/− or p18 −/− ;Brca1 +/− samples determined by the T-test.c Representative mammary tumors were immunostained with antibodies against Ck5 and Ck8.d Analysis of Ki-67-positive cells in mammary tumors.Mammary tumors developed in p18 −/− ;Gata3 +/− , p18 −/− ;Brca1 +/− , and p18 −/− mice were analyzed by IHC with an antibody against Ki-67.The percentages of Ki-67-positive cells were calculated.The results represent the mean ± SD of three individual tumors per group.e A representative mammary tumor from p18 −/− ;Gata3 +/− mouse was immunostained with GATA3.Note the heterogeneous GATA3 staining in the tumor cells.The inset shows staining of a normal-like gland in the same mouse.f Presence of the wild-type Gata3 allele in mammary tumors of p18 −/− ;Gata3 +/− mice.DNA extracted from the dissected tumor samples of mice was amplified by PCR to detect wild-type (wt) and mutant (mt) alleles of Gata3.DNA from three p18 −/− ;Gata3 +/− mammary tumors were analyzed and representative results from two tumors were shown The results represent the mean ± SD of five randomly selected fields per group.e Analysis of Gata3 levels in MMTV-PyMT (Gata3 +/+ ;Brca1 +/+ ), p18 −/− ;Gata3 +/− (Gata3 +/− ), and p18 −/− ;Brca1 +/− (Brca1 +/− ) mouse mammary tumor cells.f, g Gata3 +/+ ;Brca1 +/+ , Gata3 +/− , and Brca1 +/− tumor cells were treated with VP16 (f) for 3 h, or E2 (g) for 24 h.h, i The expression of γ-H2AX in f was quantified in h and that in g was quantified in i. j, k T47D-Sh-Ctrl, T47D-Sh-GATA3-1, and T47D-Sh-GATA3-2 cells were treated with E2 for 1 h, and the expression of genes was determined (j) and quantified (k).The blots, images, and graphs represent data from at least three independent experiments (See figure on next page.)Fig. 3 (See legend on previous page.)(Fig. 3a).We then challenged these cells with etoposide (VP16), a widely used DSB-generating agent (4,33).We noticed that 20 h after VP16 treatment the percentages of γH2AX-or 53BP1-positive cells were significantly higher in Si-GATA3 cells than in Si-control cells, indicating that depletion of WT GATA3 results in less efficient γH2AX or 53BP1 clearance and that GATA3 deficiency impairs DNA DSB repair (Fig. 3b-d, Additional file 1: Fig. S3, S4).We also determined if the treatment affects the number of T47D cells expressing cyclin A, an S-phase and cell proliferation marker.We observed that the percentages of cyclin A-positive cells were comparable in T47D-Si-GATA3 and T47D-Si-control cells treated with VP16.Notably, after VP16 treatment, some of the γH2AXfocus-positive T47D-Si-GATA3 cells co-expressed cyclin A (Fig. 3b, Additional file 1: Fig. S3b).These results also suggest that the VP16-induced higher levels of γH2AX in Gata3-deficient cells are less likely due to cell cycle arrest.In accordance with the findings derived in GATA3-deficient T47D cells, we also determined the effect of VP16 in the induction of γ-H2AX and 53BP1 in GATA3-deficient U2OS cells and obtained similar results (Additional file 1: Fig. S5).

Reconstitution of Gata3 improves DNA damage repair in Brca1-deficient mammary tumor cells and tumorigenesis
Prompted by the data that the loss of function of GATA3 generates similar phenotypes with that of BRCA1 with respect to impairing DNA damage repair (Figs. 1, 2,  3, and 4) and that GATA3 functions downstream of BRCA1 to suppress aberrant mammary cell differentiation [31,32], we then examine whether GATA3 also functions downstream of BRCA1 to control DNA repair.We took advantage of mammary tumor cell lines derived from p18 −/− ;Brca1 MGKO mice, in which both Brca1 and Gata3 were undetectable [12,32].We transduced p18 −/− ;Brca1 MGKO mammary tumor cells with pLVX-Flag or pLVX-Flag-Gata3 or transfected human MDA-MB231 cells with pBabe-Empty or pBabe-GATA3 and then treated them with ionizing radiation (IR) or VP16.We found that after IR or VP16 treatment, the level of γ-H2AX in Gata3-overexpressing cells was significantly lower than the level of γ-H2AX in control cells (Fig. 5a, Additional file 1: Fig. S6a, S6c).Although overexpression of Gata3 induced the expression of CtIP, it is noteworthy that IR or VP16 treatment did not clearly alter its expression in Gata3-overexpressing cells (Additional file 1: Fig. S6b, S6c).In addition, we also confirmed that overexpression of Gata3 did not change the expression of RNF-8 and RAD51 in response to IR or VP16 (Additional file 1: Fig. S6b, 6c).These results suggest that Gata3 protects Brca1deficient tumor cells from IR-induced DNA damage.We generated Gata3-overexpressing p18 −/− ;Brca1 MGKO mammary tumor cells and transplanted them into the MFPs of mice.We found that tumors generated by Gata3-overexpressing cells were smaller in size, as previously reported [32], and displayed significantly less γ-H2AX-positive cells than tumors generated by control cells (Fig. 5b, c, Additional file 1: Fig. S7).Notably, Gata3-overexpressing tumors expressed drastically less mesenchymal markers, Vim and Fra1, than control tumors (Fig. 5c).Taken together, these data indicate that reconstitution of Gata3 in Brca1-deficient cancer cells restores the efficiency of DNA damage repair and suppresses mesenchymal differentiation in inhibition of tumorigenesis.

GATA3 promotes HR-mediated DSB repair and restores HR efficiency of Brca1-deficient cells
DNA DSBs are repaired by two major pathways: HR and NHEJ.To directly test which pathway GATA3 is involved in DNA damage repair, we carried out assays for HR-or NHEJ-mediated repair using the DR-GFP/I-Sce I or EJ5-GFP/I-Sce I system [40].We transfected pBabe-Gata3 (Gata3) or pBabe-Empty (Empty) in HR and NHEJ reporter cell lines, which were then infected with a virus expressing I-SceI.We found that overexpression of GATA3 significantly stimulated HR by more than twofolds when compared with the HR of control cells (Fig. 6a).However, overexpression of GATA3 had no significant effect on NHEJ (Fig. 6b).In line with these results, we found that knockdown of GATA3 significantly reduced HR, but not NHEJ (Fig. 6c, d), consolidating that GATA3 promotes DNA damage repair by HR, but not NHEJ.We then focused on the role of GATA3 in BRCA1mediated HR.We found that KD of BRCA1 significantly reduced the level of HR and the expression of GATA3 (Fig. 6e), confirming not only the role of BRCA1 in promoting HR, but also our previous finding that BRCA1 regulates GATA3 in mammary cells [32].Notably, we observed that the HR efficiencies in si-BRCA1 + GATA3 and si-Control + GATA3 cells were comparable, both of which were significantly higher than the HR efficiencies in si-BRCA1 + Empty cells (Fig. 6e, left).The results indicate that reconstitution of GATA3 restores the HR efficiency of BRCA1-deficient cells.Next, we checked the role of BRCA1 in GATA3-mediated HR.We found that the HR efficiency in si-GATA3 + BRCA1 cells was significantly higher than that in si-GATA3 + Empty cells, but slightly lower than that in si-control cells (Fig. 6f, left), suggesting overexpression of BRCA1 restores HR efficiency of GATA3-deficient cells.Interestingly, we detected that the expression of GATA3 in si-GATA3 + BRCA1 cells was drastically enhanced relative to that in si-GATA3 + Empty cells (Fig. 6f, right), indicating that overexpression of BRCA1 induced GATA3 re-expression in GATA3 knockdown cells, which is likely responsible for the restoration of HR efficiency of GATA3-deficient cells.These results support that GATA3 functions downstream of BRCA1 to promote HR.

Depletion of Gata3 sensitizes tumor cells to PARP inhibitor, and reconstitution of Gata3 promotes resistance of Brca1-deficient tumor cells to PARP inhibitor
We have previously demonstrated that GATA3 is regulated by BRCA1 and GATA3 functions downstream of BRCA1 to control mammary cell differentiation [31,32].The findings that GATA3 stimulates DSB repair through HR and reconstitution of Gata3 restores the HR efficiency of Brca1-deficient cancer cells prompting us to investigate the response of Gata3-deficient tumor cells to PARP inhibitor.We treated Brca1 +/− or Gata3 +/− mammary tumor cells with olaparib (OLA), a PARP inhibitor.We noticed that the number of Brca1 +/− or Gata3 +/− tumor cells, but not the number of Gata3 +/+ ;Brca1 +/+ tumor cells, was significantly reduced after OLA treatment (Fig. 7a).In addition, OLA treatment of Gata3 knockdown tumor cells, not control cells, also resulted in a significant reduction of the cell number (Fig. 7b, c).These data support that depletion of Gata3, like that of Brca1, in mammary tumor cells impairs HR-mediated DNA DSB repair.
We ectopically overexpressed Gata3 in Brca1-deficient mammary tumor cells and then treat them with OLA.In contrast to the significant decrease in the number of Flag-expressing Brca1-deficient tumor cells, the number of Gata3-expressing Brca1-deficient tumor cells was insignificantly changed in response to OLA (Fig. 7d, e).This result indicates that reconstitution of Gata3 rescues defects of HR-mediated DNA DSBs repair in Brca1-deficient cells, which is responsible for the resistance of the cells to PARP inhibitor.
We then performed cologenic assays to further evaluate the sensitivity of the cells to PARPi and found that OLA slightly reduced the colony formation of Brca1 +/+ ;Gata3 +/+ tumor cells but significantly inhibited the colony formation of Brca1 +/− or Gata3 +/− tumor cells (Fig. 7f, Additional file 1: Fig. S8).OLA treatment of sh-Gata3-Brca1 +/+ ;Gata3 +/+ tumor cells, not sh-Ctrl-Brca1 +/+ ;Gata3 +/+ tumor cells, again significantly reduced the number of colonies formed (Fig. 7g, Additional file 1: Fig. S8).Though OLA treatment of either Flag-or Gata3-expressing Brca1-deficient tumor cells resulted in significantly less colonies than DMSO treatment, the colonies formed by Gata3-expressing cells were significantly more than those formed by Flag-expressing cells (Fig. 7h, Additional file 1: Fig. S8).These results are consistent with the data derived from the cell proliferation assay and suggest that overexpression of GATA3 partially restores PARPi resistance in BRCA1-deficient cells.

Discussion
In this study, we discovered that depletion of Gata3, like that of Brca1, impairs DNA damage repair in breast cancer cells in vitro and in vivo.Reconstitution of Gata3 improves DNA damage repair in Brca1-deficient mammary tumorigenesis.Overexpression of GATA3 promotes HR-mediated DSB repair and restores the HR efficiency of BRCA1-deficient cells.We further demonstrated that (See figure on next page.)Fig. 6 GATA3 promotes HR-mediated DNA damage repair and restores the efficiency of HR in Brca1-deficient cells.a, b The HR and NHEJ reporter cell lines, DR-GFP U2OS (a) and EJ5-GFP U2OS (b), were transfected with pBabe-Empty (Empty) or pBabe-GATA3 (GATA3), and then infected with virus expressing I-SceI.Two days later, cells were collected and analyzed for GFP expression by FACS (left panels) or for expression of GATA3 by qRT-PCR (right panels).c, d DR-GFP U2OS (c) or EJ5-GFP U2OS (d) cells were transfected with si-GATA3-1, si-GATA3-2 or si-control (si-Ctrl), infected with I-SceI, and analyzed for GFP (left) and GATA3 (right).The results in a-d represent the mean ± SD of three independent experiments.The asterisk (*) denotes a statistical significance from Empty and GATA3 or si-Ctrl and Si-GATA3 samples determined by the T-test.The number sign (#) denotes a statistical insignificance from Empty and GATA3 or si-Ctrl and Si-GATA3 samples determined by the T-test.e DR-GFP U2OS cells were firstly transfected with si-Ctrl or si-BRCA1 for 8 h and then transfected with pBabe-Empty (Empty) or pBabe-GATA3 (GATA3) for an additional 8 h.HR assay was done as above described (left) and gene expression was determined by qRT-PCR (right).f DR-GFP U2OS cells were firstly transfected with si-Ctrl or si-GATA3 for 8 h and then transfected with pBabe-Empty (Empty) or pBabe-BRCA1 (BRCA1) for an additional 8 h.HR assay was done as above described (left) and gene expression was determined by qRT-PCR (right).The results in e represent the mean ± SD of three independent  Unrepaired or improperly repaired DNA damage is responsible for genome instability that is associated with a predisposition to various types of cancer including breast cancer and grades of cancer cell differentiation [41].Inadequate DNA damage repair resulting from the depletion of BRCA1 or its interacting proteins including FANCD2, BRG1, NUMB, and HES1 promotes aberrant luminal to basal and mesenchymal differentiation in immortalized mammary epithelial cells.However, depletion of BRCA2, another BRCA1-interacting protein, impairs DNA damage repair [3,4], but does not cause aberrant differentiation, suggesting that not all BRCA1interacting proteins that functions in DNA damage repair regulate mammary cell differentiation.These discoveries prompt us to determine if transcription factors that control mammary cell differentiation play a role in DNA damage repair.
GATA3, a BRCA1-interacting transcription factor, is required for differentiation of mammary luminal epithelial cells [6,19].GATA3 is highly expressed in luminal breast cancers [23,24], but hardly detectable in BLBCs Fig. 7 Gata3-deficient tumor cells are sensitive to PARPi and reconstitution of Gata3 de-sensitizes Brca1-deficient cells to PARPi. a MMTV-PyMT (Gata3 +/+ ;Brca1 +/+ ), p18 −/− ;Gata3 +/− (Gata3 +/− ), and p18 −/− ;Brca1 +/− (Brca1 +/− ) mammary tumor cells were treated with DMSO or OLA at 10 μM.Forty-eight hours later, the cell number was counted.b, c Gata3 +/+ ;Brca1 +/+ mammary tumor cells infected with psi-LVRU6GP-control (sh-Ctrl) or psi-LVRU6GP-Gata3 (sh-Gata3), selected with puromycin, and analyzed for Gata3 expression (b).Gata3 +/+ ;Brca1 +/+ -sh-Ctrl and Gata3 +/+ ;Brca1 +/+ -sh-Gata3 cells in b were treated with DMSO or OLA for 48 h; the cell number was counted (c).d, e p18 −/− ;Brca1 +/− (Brca1 +/− ) mammary tumor cells infected with pLVX-Flag (Empty) or pLVX-Flag-Gata3 (Gata3), selected with hygromycin, and analyzed for Gata3 expression (d).Empty-and Gata3-expressing Brca1 +/− cells in (d) were treated with DMSO or OLA for 48 h; the cell number was counted (e).Data in a, c, and e represent the mean ± SD from duplicates of three independent experiments.The asterisk (*) denotes a statistical significance from DMSO-and OLA-treated samples determined by the T-test.The number sign (#) denotes a statistical insignificance from DMSO-and OLA-treated samples determined by the T-test.f-h MMTV-PyMT (Gata3 +/+ ;Brca1 +/+ ), p18 −/− ;Gata3 +/− (Gata3 +/− ), and p18 −/− ;Brca1 +/− (Brca1 +/− ) tumor cells (f), Gata3 +/+ ;Brca1 +/+ tumor cells infected with sh-Ctrl or sh-Gata3 (g), or p18 −/− ;Brca1 +/− (Brca1 +/− ) tumor cells infected with pLVX-Flag (Empty) or pLVX-Flag-Gata3 (Gata3) (h) were treated with DMSO or OLA (1 μM) and analyzed by clonogenic assay.Data represent the mean ± SD from triplicates of two independent experiments.The asterisk (*) denotes a statistical significance from DMSO-and OLA-treated samples, and the asterisk (**) denotes a statistical significance from Flag and Gata3 samples treated with OLA.The number sign (#) denotes a statistical insignificance from DMSO-and OLA-treated samples determined by the T-test [6,25].We previously reported that GATA3 functions downstream of BRCA1 to inhibit luminal to basal and mesenchymal trans-differentiation in suppression of BLBC development and progression [31,32].Not until recently has the function of GATA3 loss in regulating DNA damage repair been noticed.Depletion of GATA3 in MCF7 breast cancer cells reduces the expression of CtIP, an essential protein involved in HR, and impairs DNA damage response [33].Overexpression of WT GATA3, but not a few mutant GATA3 proteins including GATA3 D335E mutant, transactivates CtIP and promotes HR [33].Since MCF7 cells harbor a heterozygous GATA3 D335E mutant allele, they express barely detectable WT GATA3 and a large amount of mutant GATA3 [22,32,36].The finding that knockdown of GATA3 in MCF7 cells reduces CtIP expression and impairs DNA damage response [33] does not exclude the possibility that the reduced CtIP and DNA damage response resulted from the depletion of mutant, not WT, GATA3 in these cells.Therefore, the finding observed in MCF7 cells that the loss of function of GATA3 impairs DNA damage repair remains to be confirmed.In the present study, we discovered that spontaneously developed mammary tumors from p18 −/− ;Gata3 +/− mice displayed a significantly increased number of cells with DNA DSBs when compared with the tumors from p18 −/− mice.Knockdown of Gata3 in MMTV-PyMT luminal tumor cells also drastically enhanced the number of cells with DNA DSBs in tumorigenesis.Knockdown of GATA3 in human T47D (GATA3 WT) breast cancer cells or haploid loss of Gata3 by heterozygous germline deletion of Gata3 gene in mouse mammary tumor cells impaired DNA damage response in vitro.Haploid loss of Gata3 promoted estrogen-induced DNA damage in mammary tumorigenesis.Notably, as controls, in most of the above experimental systems, we noticed similar defects in DNA damage repair in Brca1-deficient tumor cells, though the grade of the defects in DNA damage repair in Gata3-deficient cells was not as strong as the grade of the defects in Brca1-deficient cells.In addition, we found that knockdown of GATA3 in T47D cells downregulates CtIP expression, confirming that GATA3 positively regulates the expression of CtIP.Together, our data demonstrate that depletion of GATA3 impairs DNA damage response in mammary tumor cells in vitro and in mammary tumorigenesis in vivo.
Interaction among BRCA1, GATA3, and CtIP in DNA damage repair and mammary cell differentiation is quite interesting.GATA3 stimulates the expression of CtIP and GATA3 itself is regulated by BRCA1 [32,33].Both GATA3 and CtIP are BRCA1-interacting proteins [1,19,42].BRCA1-CtIP complex plays a critical/central role in HR-mediated DNA DSB repair [42], while BRCA1-GATA3 complex protects mammary cells from aberrant differentiation [12,19,32].Interestingly, heterozygous germline deletion and mammary epithelial cellspecific deletion of CtIP do not result in the spontaneous development of mammary tumors, and loss of CtIP function may inhibit mammary tumor formation in p53-deficient mice [43].However, since the gene targeting of CtIP is not confirmed [43] and the mammary tumors developed in p53-deficient mice are typical BLBCs with EMT features [44], it remains elusive whether CtIP plays a role in regulating luminal to basal or mesenchymal differentiation in normal or cancerous mammary cells.We previously discovered that Brca1 positively regulates GATA3 expression and that GATA3 functions downstream of BRCA1 suppressing luminal-to-basal and to mesenchymal differentiation in mammary cells as well as tumor development and progression [31,32].We found in this study that overexpression of GATA3 significantly promotes HR.Reconstitution of Gata3 in Brca1-deficient mammary tumor cells restores the HR efficiency of Brca1-deficient cells and improves DNA damage repair in mammary tumorigenesis.Depletion of Gata3, similar to Brca1 depletion, sensitizes tumor cells to PARP inhibitor-induced cell death.Reconstitution of Gata3 enhances resistance of Brca1-deficient tumor cells to PARP inhibitor.Taking into consideration our previous discovery that Brca1 positively regulates GATA3 expression and GATA3 functions downstream of BRCA1 suppressing luminalto-basal and mesenchymal differentiation in mammary cells as well as tumor development and progression [31,32], our findings demonstrate that GATA3 functions downstream of BRCA1 to promote HR-mediated DNA damage repair and to suppress aberrant differentiation in mammary and tumor development.

Conclusions
These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression.Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs.

Mammary tumor cell preparation, transplantation, and tumor treatment
Mammary tumors were dissected from female mice and tumor cell suspensions were prepared as previously described [12,25,35].For mammary tumor cell transplantation, cells were suspended in a 50% solution of Matrigel (BD) and then inoculated into the left and/or right inguinal mammary fat pads (MFPs) of 4-6-weekold NSG or NCG mice, respectively.Six or 7 weeks after transplantation, animals were euthanized and mammary tumors were dissected for histopathological, immunohistochemical, and biochemical analyses.For estrogen treatment in vivo, 0.72 mg E2 (SE-121, IRA, Sarasota, FL, USA) or Beeswax pellet was implanted subcutaneously in mice receiving tumor cell or tissue transplants.

HR and NHEJ repair assays
The green fluorescent protein (GFP) reporter system for HR-mediated DSB repair (DR-GFP U2OS cells), the GFP reporter system for NHEJ-mediated DSB repair (EJ5-GFP U2OS cells), and the I-SceI expression construct were generous gifts from Maria Jasin (Memorial Sloan Kettering Cancer Center).HR assay was performed as described [40].Briefly, DR-GFP U2OS cells were transfected with pBabe-GATA3, pBabe-BRCA1, pBabe-Empty, or siRNA oligonucleotide duplexes for 8 h, and 8 h later infected with virus expressing I-SceI.DR-GFP U2OS cells were transfected with siRNA oligonucleotide duplexes for 8 h, then transfected with pBabe-GATA3, pBabe-BRCA1, or pBabe-Empty for 8 h, and lastly, infected with virus expressing I-SceI.Two days after infection by I-SceI, the fraction of GFP-positive cells was determined by FACS.NHEJ assay was done as described [49].Briefly, EJ5-GFP U2OS cells were transfected with pBabe-GATA3, pBabe-Empty, or siRNA oligonucleotide duplexes for 8 h.Eight hours later, the cells were infected with I-SceI.Two days later, GFP-positive cells were determined by FACS.

Statistical analysis
All data are presented as the mean ± SD for at least three repeated individual experiments for each group.Sample sizes, number of replicates, and normalization methods are indicated in each figure legend.No statistical methods were used to pre-determine sample/group sizes.Statistical analyses were performed as described in each figure legend using GraphPad PRISM 6.02 software.Quantitative results were analyzed by two-tailed Student's t test.p < 0.05 was considered statistically significant.

Fig. 5
Fig. 5 Reconstitution of GATA3 in BRCA1-deficient tumor cells reduces DNA damage.a p18 −/− ;Brca1 MGKO mammary tumor cells infected with pLVX-Flag (Empty) or pLVX-Flag-Gata3 (Gata3) were treated with or without ionizing radiation (IR) of 5 Gy, and the expression of Gata3 and γ-H2AX were analyzed at different time points after IR treatment.b, c p18 −/− ;Brca1 MGKO mammary tumor cells infected with infected with pBabe-puro-Gata3 (Gata3) and pBabe-puro-empty (Empty) were transplanted into left and right inguinal MFP of mice.Four weeks later, the regenerated mammary tumors were analyzed by IHC with antibodies against γH2AX (b) Gata3, Fra1, or Vim (c).The H-scores for γH2AX in b were calculated.The results represent the mean ± SD of three individual tumors per group.The asterisk (*) denotes a statistical significance from empty-and Gata3-overexpressed samples determined by the T-test.The blots and images represent data from at least three independent experiments or tumors experiments.The asterisk (*) denotes a statistical significance from Si-Ctrl + Empty and Si-Ctrl + GATA3, Si-BRCA1 + Empty, or Si-BRCA1 + GATA3 samples determined by the T-test.The asterisk (**) denotes a statistical significance from Si-BRCA1 + Empty and Si-BRCA1 + GATA3 samples determined by the T-test.The number sign ( # ) denotes a statistical insignificance from Si-Ctrl + Empty and Si-Ctrl + GATA3 samples determined by the T-test.The results in f represent the mean ± SD of three independent experiments.The asterisk (*) denotes a statistical significance from Si-Ctrl and Si-GATA3 + Empty samples determined by the T-test.The asterisk (**) denotes a statistical significance from Si-GATA3 + Empty and Si-GATA3 + BRCA1 samples determined by the T-test.The number sign ( # ) denotes a statistical insignificance from Si-Ctrl and Si-GATA3 + BRCA1 samples determined by the T-test depletion of Gata3 sensitizes tumor cells to PARP inhibitor, and reconstitution of Gata3 enhances resistance of Brca1-deficient tumor cells to PARP inhibitor.Together with our previous findings, these results demonstrate that GATA3 functions downstream of BRCA1 controlling HR-mediated DNA damage repair and aberrant